Emilia M. F. Mauriello

Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA.

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S‐motility powered by type‐IV pili and A‐motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin‐like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S‐ and A‐motility, causing rapid dispersal of S‐ and A‐motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a ΔmglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA–YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A‐motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin‐like protein and a small GTPase spatial regulator.

Gliding Motility Revisited: How Do the Myxobacteria Move without Flagella?

SUMMARY In bacteria, motility is important for a wide variety of biological functions such as virulence, fruiting body formation, and biofilm formation. While most bacteria move by using specialized appendages, usually external or periplasmic flagella, some bacteria use other mechanisms for their movements that are less well characterized. These mechanisms do not always exhibit obvious motility structures. Myxococcus xanthus is a motile bacterium that does not produce flagella but glides slowly over solid surfaces. How M. xanthus moves has remained a puzzle that has challenged microbiologists for over 50 years. Fortunately, recent advances in the analysis of motility mutants, bioinformatics, and protein localization have revealed likely mechanisms for the two M. xanthus motility systems. These results are summarized in this review.

A multi-protein complex from Myxococcus xanthus required for bacterial gliding motility

Myxococcus xanthus moves by gliding motility powered by Type IV pili (S‐motility) and a second motility system, A‐motility, whose mechanism remains elusive despite the identification of ∼40 A‐motility genes. In this study, we used biochemistry and cell biology analyses to identify multi‐protein complexes associated with A‐motility. Previously, we showed that the N‐terminal domain of FrzCD, the receptor for the frizzy chemosensory pathway, interacts with two A‐motility proteins, AglZ and AgmU. Here we characterized AgmU, a protein that localized to both the periplasm and cytoplasm. On firm surfaces, AgmU‐mCherry colocalized with AglZ as distributed clusters that remained fixed with respect to the substratum as cells moved forward. Cluster formation was favoured by hard surfaces where A‐motility is favoured. In contrast, AgmU‐mCherry clusters were not observed on soft agar surfaces or when cells were in large groups, conditions that favour S‐motility. Using glutathione‐S‐transferase affinity chromatography, AgmU was found to interact either directly or indirectly with multiple A‐motility proteins including AglZ, AglT, AgmK, AgmX, AglW and CglB. These proteins, important for the correct localization of AgmU and AglZ, appear to be organized as a motility complex, spanning the cytoplasm, inner membrane and the periplasm. Identification of this complex may be important for uncovering the mechanism of A‐motility.

Localization of a bacterial cytoplasmic receptor is dynamic and changes with cell-cell contacts

Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor, does not form membrane-bound polar clusters typical for most bacteria, but rather cytoplasmic clusters that appear helically arranged and span the cell length. The distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters that continuously changed their size, number, and position. The number of FrzCD clusters was correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing mutants and additional clusters were observed in hyper-reversing mutants. When moving cells made side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These events were frequently followed by one of the interacting cells reversing. These observations suggest that FrzCD detects signals from a cell contact-sensitive signaling system and then re-localizes as it directs reversals to distributed motility engines.

AglZ regulates adventurous (A-) motility in Myxococcus xanthus through its interaction with the cytoplasmic receptor, FrzCD

Myxococcus xanthus moves by gliding motility powered by type IV pili (S‐motility) and distributed motor complexes (A‐motility). The Frz chemosensory pathway controls reversals for both motility systems. However, it is unclear how the Frz pathway can communicate with these different systems. In this article, we show that FrzCD, the Frz pathway receptor, interacts with AglZ, a protein associated with A‐motility. Affinity chromatography and cross‐linking experiments showed that the FrzCD–AglZ interaction occurs between the uncharacterized N‐terminal region of FrzCD and the N‐terminal pseudo‐receiver domain of AglZ. Fluorescence microscopy showed AglZ–mCherry and FrzCD–GFP localized in clusters that occupy different positions in cells. To study the role of the Frz system in the regulation of A‐motility, we constructed aglZ frzCD double mutants and aglZ frzCD pilA triple mutants. To our surprise, these mutants, predicted to show no A‐motility (A‐S+) or no motility at all (A‐S‐), respectively, showed restored A‐motility. These results indicate that AglZ modulates a FrzCD activity that inhibits A‐motility. We hypothesize that AglZ–FrzCD interactions are favoured when cells are isolated and moving by A‐motility and inhibited when S‐motility predominates and A‐motility is reduced.

A Versatile Class of Cell Surface Directional Motors Gives Rise to Gliding Motility and Sporulation in Myxococcus xanthus

The Myxococcus Agl-Nfs machinery, a type of bacterial transport system, is modular and is seen to also rotate a carbohydrate polymer directionally at the spore surface to assist spore coat assembly.

Functional organization of a multimodular bacterial chemosensory apparatus.

Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium Myxococcus xanthus, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. M. xanthus contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on M. xanthus social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, in vivo, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.

Evolution and Design Governing Signal Precision and Amplification in a Bacterial Chemosensory Pathway.

Understanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A- and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways.

Chemotaxis mediated by NarX–FrzCD chimeras and nonadapting repellent responses in Myxococcus xanthus

Myxococcus xanthus requires gliding motility for swarming and fruiting body formation. It uses the Frz chemosensory pathway to regulate cell reversals. FrzCD is a cytoplasmic chemoreceptor required for sensing effectors for this pathway. NarX is a transmembrane sensor for nitrate from Escherichia coli. In this study, two NarX–FrzCD chimeras were constructed to investigate M. xanthus chemotaxis: NazDF contains the N‐terminal sensory module of NarX fused to the C‐terminal signalling domain of FrzCD; NazDR is similar except that it contains a G51R mutation in the NarX domain known to reverse the signalling output of a NarX–Tar chimera to nitrate. We report that while nitrate had no effect on the wild type, it decreased the reversal frequency of M. xanthus expressing NazDF and increased that of M. xanthus expressing NazDR. These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazDR strain failed to adapt to nitrate in temporal assays as did the wild type to known repellents. The lack of temporal adaptation to negative stimuli appears to be a general feature in M. xanthus chemotaxis. Thus, the appearance of biased movements by M. xanthus in repellent gradients is likely due to the inhibition of net translocation by repellents.

Cell polarity/motility in bacteria: closer to eukaryotes than expected?

The Gram‐negative bacterium Myxococcus xanthus glides on solid surfaces and periodically reverses the direction of movement. Work published in this issue of The EMBO Journal (Leonardy et al , 2010) reports on the small GTPase MglA that ensures the correct polarity of the motility engines through its GTP/GDP cycle in conjunction with its cognate GAP, MglB. MglA has also been shown to interact with the actin‐like protein MreB in eukaryotic‐like motility complexes. Altogether, the data suggest that compelling similarities exist between the mechanisms of motility and establishment of cell polarity in M. xanthus and eukaryotes.